Naoyuki Iida

Requirement of Ras for the Activation of Mitogen-Activated Protein Kinase by Calcium Influx, cAMP, and Neurotrophin in Hippocampal Neurons

Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitroand in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1m, or dominant negative Ras by means of adenovirus vectors. Gap1m expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNF-induced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection

Salmonella phosphothreonine lyase SpvC inactivates the dual‐phosphorylated host mitogen‐activated protein kinases (MAPK) through β‐elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)‐1 and SPI‐2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI‐2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI‐1 and SPI‐2 T3SSs. Dephosphorylation of the extracellular signal‐regulated protein kinases (ERK) was detected in an SPI‐1 T3SS‐dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild‐type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro‐inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti‐inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.

I2020T leucine-rich repeat kinase 2, the causative mutant molecule of familial Parkinson’s disease, has a higher intracellular degradation rate than the wild-type molecule

Leucine-rich repeat kinase 2 (LRRK2) has been identified as the causal gene for autosomal dominant familial Parkinsons disease (PD), although the mechanism of neurodegeneration involving the mutant LRRK2 molecules remains unknown. In the present study, we found that the protein level of transfected I(2020)T mutant LRRK2 was significantly lower than that of wild-type and G(2019)S mutant LRRK2, although the intracellular localization of the I(2020)T and wild-type molecules did not differ. Pulse-chase experiments proved that the I(2020)T LRRK2 molecule has a higher degradation rate than wild-type or G(2019)S LRRK2. Upon addition of proteasome and lysosome inhibitors, the protein level of I(2020)T mutant LRRK2 reached that of the wild-type. These results indicate that I(2020)T mutant LRRK2 is more susceptible to post-translational degradation than the wild-type molecule. Our results indicate a novel molecular feature characteristic to I(2020)T LRRK2, and provide a new insight into the mechanism of neurodegeneration caused by LRRK2.

Aralin, a type II ribosome-inactivating protein from Aralia elata, exhibits selective anticancer activity through the processed form of a 110-kDa high-density lipoprotein-binding protein: a promising anticancer drug.

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the 110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors.

Identification of protein kinase substrates by proteomic approaches

This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.

Effective Heat therapy Controlling Heat Deposition of Microbubbles in the Ultrasound Field

It is required that the tumor site is coagulated less invasively and more efficiently, and HIFU treatment combined with microbubbles is investigated here. Microbubbles have a potential to enhance the heating effect in the ultrasound field. In this study, the relationship between the thermal effect and the behavior of microbubbles is analyzed. The temperature rise at the focal region was measured by a thermocouple, and the bubble behavior was simultaneously observed with a high‐speed camera. The ultrasound frequency was 2.2 MHz and the intensity was 1000 – 5000 W/cm2. This time Levovist®, one of the contrast agents, was used as microbubbles, and the initial void fraction (the ratio of total gas volume to liquid) around the focal region was 0, 10−5, 10−4, 10−3 and 10−2%. The temperature rise became larger as the intensity of ultrasound and the void fraction increased. Comparing the data of the thermocouple with the photographs taken by the camera, the temperature rise became large as the bubbles were violen...

Proteomic identification of p38 MAP kinase substrates using in vitro phosphorylation

Protein phosphorylation is a major mechanism that regulates many basic cellular processes. Identification and characterization of substrates for a given protein kinase can lead to a better understanding of signal transduction pathways. However, it is still difficult to efficiently identify substrates for protein kinases. Here, we propose an integrated proteomic approach consisting of in vitro dephosphorylation and phosphorylation, phosphoprotein enrichment, and 2D‐DIGE. Phosphatase treatment significantly reduced the complexity of the phosphoproteome, which enabled us to efficiently identify the substrates. We employed p38 mitogen‐activated protein kinase (p38 MAP kinase) as a model kinase and identified 23 novel candidate substrates for this kinase. Seven selected candidates were phosphorylated by p38 MAP kinase in vitro and in p38 MAP kinase‐activated cells. This proteomic approach can be applied to any protein kinase, allowing global identification of novel substrates.

Development of HIFU treatment in which the heating location is controlled using microbubbles

High‐intensity focused ultrasound (HIFU) treatment that employs microbubbles to provide enhanced heating has been investigated in order to develop a less invasive and more rapid tumor therapy. Previous studies by us have demonstrated that ultrasound propagation is disturbed when there are microbubbles in front of the focus. In this study, we develop a method for obtaining enhanced heating by using microbubbles just at the focus, thus avoiding heating on the transducer side. In this method, microbubbles are destroyed in front of the HIFU focus (on the transducer side) by irradiating a very short burst wave of microsecond order, before irradiating the ultrasound waves for heating the focus. The experiment is conducted in a medium of a gel containing microbubbles, and a temperature‐sensing liquid crystal sheet is set in the focus to observe the temperature distribution. The ultrasound frequency was 2.2 MHz and the intensity was 5000 W/cm2, and 20 burst wave waves were irradiated at pulse repetition frequency...

Thermal effect of microbubbles in the focused ultrasound field

High‐intensity focused ultrasound (HIFU) has been developed for the treatment of tumor, and additionally the medical applications with microbubbles such as ultrasound imaging have attracted much attention. In the field of the bubble dynamics, it is known that the microbubble is an energy converter of ultrasound mechanical energy to heat when subjected to an acoustic field. The goal is the tissue heating enhanced by microbubbles. In this study, the relationship between the heating effect and the behavior of microbubbles is analyzed. The temperature rise was measured by a thermocouple and a thermal liquid crystal sheet, and the bubble behavior was simultaneously observed with a camera. The ultrasound frequency is 2.2 MHz and the intensity is 100–2000 W/cm2. The temperature rise became larger as the number density of microbubbles (Levovist) around the focal region increased. As the number density became too large, the conversion efficiency from ultrasound energy to heat became smaller because the shielding e...